MDA in the colon was extracted and measured by high-performance liquid chromatography-fluorescence detection (HPLC-FLD) as previously described with minor modifications [32 (link)]. Briefly, 50 mg of colon was homogenized in 0.5 mL ice-cold 0.15 mol/L KCl containing 0.01% BHT using a Fisher Scientific™ Bead Mill 4 homogenizer (Pittsburgh, PA, USA). Then, 50 µL homogenate was mixed with 400 µL of KCl, 40 µL of 0.2% BHT in ethanol and 200 µL of 1N NaOH, and then incubated at 60 °C for 30 min. Then, protein was precipitated by mixing with 2 mL of 5% TCA. After centrifugation at 1000× g for 10 min at 4 °C, 500 µL of supernatant was derivatized with 0.6% TBA at 95 °C for 30 min. Then, the sample was extracted with butanol, and injected onto a Dionex UltiMate 3000 HPLC equipped with an LPG-3400 quaternary pump, a WPS-3000 analytical autosampler, and an FLD-3100 fluorescence detector (Thermo Fisher Scientific, San Jose, CA, USA). Samples were resolved isocratically at 0.8 mL/min on a Discovery® C18 column (250 × 4.6 mm, 5µm; Supelco, Bellefonte, PA, USA) using 40:60 methanol and 25 mM potassium phosphate buffer (pH 6.5). MDA was quantified against standards prepared in parallel from TMP and was normalized to colonic protein.
Bead mill 4 homogenizer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bead Mill 4 homogenizer is a laboratory equipment designed for the efficient mechanical disruption of biological samples. It utilizes high-speed agitation of sample-containing tubes or vials filled with beads to effectively break down and homogenize a variety of materials, including cells, tissues, and other solid samples.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Fld 3100 fluorescence detector, by Thermo Fisher Scientific (1 mentions)
Discovery c18 column, by Merck Group (1 mentions)
Ceramic beads, by Thermo Fisher Scientific (1 mentions)
Benzonase endonuclease, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Nanodrop, by Mettler Toledo (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Primerquest tool, by Integrated DNA Technologies (1 mentions)
Sybr green, by Qiagen (1 mentions)
Iscripttm cdna synthesis kit, by Bio-Rad (1 mentions)
Direct zoltm rna miniprep kit, by Zymo Research (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
12 protocols using bead mill 4 homogenizer
1
Colon MDA Quantification via HPLC-FLD
2
Extraction and Preparation of Frozen Brain Tissue Samples
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Frozen brain tissue stored at −8°C was mechanically pulverized using a stainless-steel mortar and pestle on dry ice. We then added 600 μL of ice-cold lysis buffer [20 mm Tris, 1 mm EDTA, 1 mm EGTA, 240 mm sucrose, 1× Halt Protease and Phosphatase Inhibitor (ThermoFisher)] to 1.5-ml tubes containing ceramic beads (#15-340-153, Fisherbrand), followed by 100 mg of pulverized frozen brain. We homogenized the samples using a Bead Mill 4 homogenizer (Fisherbrand) with velocity 5 m/s for a total of 20 s. We removed the supernatant and transferred it into a sterile low-protein binding microcentrifuge tube, spun at 15,000 × g for 30 min at 4 C, and the supernatant then discarded. We measured final protein concentrations in the pellet using a BCA (bicinchoninic acid) assay before proceeding directly to immunoprecipitation (IP) or storage at −80° C. Samples were prepared for co-immunoprecipitation through treatment with Benzonase endonuclease (#9025-65-4, Millipore) for 1 μg per reaction and 15-min room temperature immediately before co-IP. We used benzonase because in our pilot study, samples without benzonase were extremely viscous, raising concerns for nonspecific interactions in the resulting mass spectrometry data (data not shown).
3
Extraction and Quantification of IR-780 Dye from Tumor Tissue
4
Measuring Glutathione Peroxidase Activity
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Colon samples were rinsed with ice-cold PBS and then homogenized in 5 mL of ice-cold PBS (with 5 mM EDTA and 1 mM DTT) per g tissue with a Fisher Scientific™ Bead Mill 4 homogenizer (Pittsburgh, PA, USA). The homogenate was then centrifuged at 10,000× g for 15 min at 4 °C. Supernatant aliquots were used to measure GPx activity by an assay kit (Cayman Chemical, Ann Arbor, MI) and to determine protein content by the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). GPx activity was expressed as nmol/min/µg protein.
5
Virus Purification from Coracidia
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Coracidia from S. solidus families from Wolf Lake, Loberg Lake, and Cheney Lake were used for virus purification before conducting a second sequencing effort (Table S1 ). PCR assays were used to select families infected by viruses (see below). Coracidia were homogenized in sterile suspension medium buffer through bead beating in a Fisherbrand Bead Mill 4 Homogenizer for 1 min using a mixture of glass beads. Virus particles were purified from homogenates through 0.45 μm filtration and nuclease treatment following methods used to characterize virions isolated from arthropods [29 (link)].
6
Quantitative Gene Expression Analysis
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Total RNA was extracted from samples using the RNeasy Mini Kit (Qiagen). Cells were lysed directly in their culture wells and tissues were lysed and homogenized using the Bead Mill 4 Homogenizer (Fisher Scientific). Once extracted, RNA was quantified using a NanoDrop (Mettler Toledo), with a concentration of at least 10 ng/μl being required to proceed with reverse transcription. cDNA was synthesized using the Quantitect Reverse Transcription Kit (Qiagen) and stored at -20 °C for further use. Primers for qRT-PCR were designed using the Primer Quest tool (Integrated DNA Technologies) and SYBR Green (Qiagen) was used as the DNA intercalating dye. qRT-PCR plates were run using the QuantStudio 5 Real-Time PCR system (Applied Biosystems) with a total reaction volume of 20 µL. Expression levels of genes of interest were normalized to HPRT1 levels and fold change values were obtained using the 2−ΔΔCT method. At least three to five independent samples were run for each gene expression assay.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from a section of the left lung lobe using Trizol (Invitrogen, Carlsbad, CA, United States) and two runs of a Bead Mill 4 homogenizer (Fisher Scientific, Newington, NH, United States) at a speed of 5 m/s (500 Watts) for 30 s. Afterward, total RNA was extracted and purified utilizing Direct-zolTM RNA MiniPrep Kits (Zymo Research) as per the manufacturer’s instructions. RNA concentration was quantified and quality assessed using a Nanodrop (Thermo Scientific, Hercules, CA, United States). An aliquot of 1 μg of RNA was reverse transcribed into cDNA using an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, United States) as per the manufacturer’s instructions. Quantitative real time RT-PCR analysis was performed using inventoried primers (Qiagen, Hilden, Germany) to evaluate altered gene expression of interleukin-6 (IL-6), interleukin 1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), chemokine ligand 1 (CXCL1), arachidonate 5-lipoxygenase (ALOX-5), arachidonate 15-lipoxygenase (ALOX-15), and inducible nitric oxide synthase (iNOS). In all cases, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Fold changes were calculated comparing all sample values individually to the average of the unexposed control healthy mouse model.
8
Tissue Homogenization and Extraction
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Kidney, retina, and brain tissues were weighed and homogenized at a density of 140 mg of dry tissue per milliliter in ethyl acetate/isopropanol (4:1) using a Bead Mill 4 homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) and 2 mL pre-filled polypropylene microtubes (2.8 mm ceramic beads, 3 × 20 s cycles, speed 5). Insoluble debris was removed by centrifugation (10,000× g, 30 min, 5 °C). Fixed aliquots of the supernatants (225 μL) were transferred to clean Eppendorf tubes and evaporated under a gentle stream of nitrogen gas. The residues were reconstituted in 75 μL of methanol/water (4:1), centrifuged (18,000× g, 30 min, 5 °C), and transferred to 2 mL autosampler vials equipped with low-volume polypropylene inserts and Teflon-lined rubber septa for analysis.
9
Tissue Preparation for Metabolomic Analysis
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Kidney, retina, and brain tissues were weighed and homogenized at a density of 140 mg of dry tissue per milliliter in ethyl acetate / isopropanol (4:1) using a Bead Mill 4 homogenizer (Thermo Fisher Scientific, Waltham MA) and 2-mL pre-filled polypropylene microtubes (2.8 mm ceramic beads, 3 × 20 sec cycles, speed 5). Insoluble debris was removed by centrifugation (10,000 × g, 30 min, 5 °C). Fixed aliquots of the supernatants (225 μL) were transferred to clean Eppendorf tubes and evaporated under a gentle stream of nitrogen gas. The residues were reconstituted in 75 μL of methanol/water (4:1), centrifuged (18,000 × g, 30 min, 5 °C), and transferred to 2-mL autosampler vials equipped with low-volume polypropylene inserts and Teflon-lined rubber septa for analysis.
10
Quadriceps Protein Extraction and Western Blotting
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The quadriceps were homogenized in 60 µL/mg ice-cold RIPA buffer (10 mM Tris–HCl pH 7.4, 5 mM EDTA, 0.1% SDS, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton) with 1 mM phenylmethanesulfonyl fluoride and a protease/phosphatase inhibitor cocktail (#5872, Cell Signaling, Danvers, MA, USA) using a Bead Mill 4 Homogenizer (Thermo Fisher Scientific, Waltham, MA, USA), and then centrifuged (15,000× g for 10 min at 4 °C). The supernatant protein concentration was determined with Bradford Reagent (Sigma, Burlington, VT, USA) using a microplate reader (Tristar 5, Berthold, Bad Wildbad, Germany). Forty micrograms of denatured proteins were separated in SDS polyacrylamide 12% and transferred onto polyvinylidene fluoride membranes according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). After overnight incubation with primary antibodies and then horseradish peroxidase-conjugated secondary antibodies (see Table S3 for details), the protein complexes were revealed (Kit Clarity Western ECL substrate, Bio-Rad, Hercules, CA, USA) using chemiluminescence (Fusion X Spectra, Vilber, Marne-la-Vallée, France). The protein expression was quantified with ImageJ software (NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/ ) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GADPH).
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